The thioredoxin system is a major cellular redox system that is present in all previously characterized organisms. Thioredoxin reductases control the redox state of thioredoxins, which in turn regulate numerous cellular processes. Thioredoxin reductases have also been implicated in the redox control of other cellular proteins and compounds. The function and catalytic activity of mammalian thioredoxin reductases is dependent on a selenocysteine residue, whose biosynthesis in turn is affected by the availability of dietary selenium. Mammals contain three selenoprotein thioredoxin reductase isozymes, TR1, TR3 and TGR, which occur in multiple forms due to alternative splicing. The purpose of the proposed study is to functionally characterize mammalian thioredoxin reductases, with emphasis on the specific roles of isozymes and their alternative forms in redox regulation of cellular processes. A combination of biochemical, cell biology and mouse model approaches will be used to address the following specific questions (specific aims): (i) what is the specific role of TGR in male reproduction? We will test a hypothesis that this enzyme isomerizes disulfide bonds in proteins. In addition, a knockout mouse model will be developed to directly test the requirement of this protein for sperm maturation, (ii) what are the roles of specific isoforms of thioredoxin reductases? We will characterize the roles of mitochondrial and cytosolic forms of TR3 and identify targets of all three thioredoxin reductases. We will also test a hypothesis that TGR generates a mitochondrial form from a non-canonical initiation codon. (iii) What are the determinants for preferential supply of thioredoxin reductases with selenium? We will examine elements in thioredoxin reductase genes responsible for this effect.